Lactobacillus plantarum, fermented chrysanthemum, their preparation methods and applications in improving bone health

Abstract

This invention provides a strain of *Lactobacillus plantarum*, and discloses the fermentation product of *Eucommia ulmoides* leaves, *Rehmannia glutinosa*, dried ginger, and *Dendrobium officinale* obtained by fermenting these herbs, along with its preparation method and applications. This fermentation product can directionally activate the phenylpropane biosynthetic pathway, inducing the generation of characteristic small molecule metabolites of polyphenols, particularly sanguranyl sulfadiazine (SSA). Moreover, compared to the raw materials, the fermentation product contains more active small molecule metabolites. Finally, experiments on zebrafish modeled with prednisone demonstrated that the fermentation product can significantly promote bone formation. The fermentation product mainly restores the expression levels of related genes to normal zebrafish gene expression levels by regulating these genes. Therefore, the fermentation product can be used to prepare foods or medicines that improve bone health.

Description

Technical Field

[0001] This invention relates to the field of probiotics, specifically to a plant lactobacillus, and the fermentation product of Eucommia ulmoides leaves, Rehmannia glutinosa, dried ginger and Dendrobium officinale obtained by fermenting Eucommia ulmoides leaves, Rehmannia glutinosa, dried ginger and Dendrobium officinale, the preparation method thereof and its application in bone health. Background Technology

[0002] As the core tissue supporting human movement, regulating metabolism, and providing the hematopoietic microenvironment, the skeleton undertakes multiple functions, including mechanical support, calcium and phosphorus homeostasis regulation, and bone marrow hematopoiesis. It plays an irreplaceable role in maintaining physical activity, metabolic balance, and the homeostasis of life and health.

[0003] Osteoarthritis and osteoporosis are common degenerative bone diseases among middle-aged and elderly people. Long-term illness not only induces chronic pain and loss of limb function, but also increases the risk of complications such as cardiovascular events and depression, placing a heavy burden on public health resources. Prevention and treatment are urgent.

[0004] While commonly used anti-osteoporosis drugs (such as bisphosphonates) can slow bone loss, they pose risks such as gastrointestinal irritation and mandibular osteonecrosis. Symptomatic treatment for osteoarthritis (such as nonsteroidal anti-inflammatory drugs) can only relieve symptoms and cannot reverse cartilage damage; long-term use can also lead to liver and kidney damage. Therefore, finding safe, long-acting, and reversible interventions is a key challenge in the prevention and treatment of degenerative bone diseases.

[0005] This invention employs a fermentation method based on the homology of food and medicine. Traditional Chinese medicine theory holds that "the kidney governs bones and produces marrow." The ancient formula for strengthening bones and tendons recorded in *Qianjin Fang* (Prescriptions Worth a Thousand Gold Pieces) uses Eucommia ulmoides leaves, Rehmannia glutinosa, Dendrobium nobile, and dried ginger as food and medicinal materials, possessing the effects of "tonifying the liver and kidneys and strengthening tendons and bones." Modern research confirms that through microbial fermentation of food and medicinal materials, the transformation and enrichment of active substances can be achieved through the metabolism of microbial communities: Firstly, the fermentation process can degrade macromolecular components in raw materials, generating more easily absorbed metabolites such as phenolic acids (e.g., chlorogenic acid) and flavonoids (e.g., quercetin), thus improving their bioavailability. Secondly, fermentation can improve flavor and remove unpleasant flavors from traditional Chinese medicine. Thirdly, fermentation can degrade toxins and reduce anti-nutritional factors. However, most current products and patents focus on enhancing the efficacy of active ingredients and improving bioavailability, neglecting the aspect of detoxification and efficacy enhancement. Therefore, this invention develops a food-medicine fermentation product that combines food safety and pharmacological activity. It can achieve the goal of "food and medicine from the same source" in the treatment of chronic bone diseases, while avoiding the risk of toxic side effects of chemical drugs. It provides a green and sustainable new strategy for the prevention and treatment of bone health (osteoporosis, osteoarthritis). Summary of the Invention

[0006] The first objective of this invention is to provide a strain of *Lactobacillus plantarum* YS. GuLin18.

[0007] The second objective of this invention is to provide a fermented product obtained by fermenting the combination of Eucommia ulmoides leaves, Rehmannia glutinosa, dried ginger, and Dendrobium officinale with the probiotics.

[0008] The third objective of this invention is to provide a method for preparing the fermented product of *Zhonghuang*.

[0009] A fourth objective of this invention is to provide an application of the aforementioned fermented turmeric.

[0010] This invention is achieved through the following technical solution:

[0011] A plant lactobacillus (Lactiplantibacillus plantarum) YS GuLin18, with accession number CGMCC NO. 35529, was deposited on August 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0012] Institute of Microbiology, Chinese Academy of Sciences, Postcode 100101, Accession number CGMCC NO. 35529. This bacterium was viable at the time of preservation.

[0013] A fermented product containing *Lactobacillus plantarum* YS GuLin18 fermentation is obtained from a food and medicine homology composition consisting of 70wt% Eucommia ulmoides leaves, 15wt% Rehmannia glutinosa, 10wt% dried ginger, and 5wt% Dendrobium officinale.

[0014] The preparation method of the fermented product of Rheum palmatum includes the following steps:

[0015] Lactobacillus plantarum YS GuLin18 seed culture was inoculated into a sterilized substrate consisting of glucose, peptone, dipotassium hydrogen phosphate, a food-medicine homologous composition, and water. β-carotene was then added to the substrate. The first fermentation step was carried out by dextran hydrolase, followed by the addition of vitamin C to the substrate for the second fermentation step, then the addition of quinic acid to the substrate for the third fermentation step, and finally inactivation, centrifugation, and collection of the supernatant.

[0016] The method for preparing the seed liquid includes the following steps: [The text abruptly shifts to a seemingly unrelated topic about Lactobacillus plantarum and its preparation.] GuLin18 was inoculated into MRS liquid medium and cultured to obtain a bacterial suspension. Then, 2% of the bacterial suspension was inoculated into MRS liquid medium to obtain a seed culture. The seed culture contained *Lactobacillus plantarum* YS... The concentration of GuLin18 is 1×10 9 CFU / mL.

[0017] The substrate includes 4 6% glucose, 0.5 1% peptone, 0.10 0.25% dipotassium hydrogen phosphate, 6 8% Food and drug homology composition.

[0018] The sterilization temperature is 115℃ and the time is 20 min; the inoculation amount of the seed liquid is 2%.

[0019] The β The activity of the dextran hydrolase is 20 U / g, and the fermentation temperature of the first fermentation step is 30°C. 32℃; the amount of vitamin C added is 0.1%, and the fermentation temperature for the second fermentation step is 35℃. 37℃; the amount of quinic acid added is 0.05%, and the fermentation temperature of the third fermentation step is 35℃. 37℃.

[0020] The final pH value of the third fermentation step was 3.8. 4.0; the inactivation temperature is 115℃, and the time is 30 min; the centrifugation speed is 6000 r / min, and the time is 3... 5 minutes.

[0021] The aforementioned fermented ginseng can be used to prepare drugs that increase the area of ​​the skull; or to prepare drugs that improve bone health by downregulating the expression levels of runx2a and / or acp5a.

[0022] The aforementioned fermented cyperus extract is used in drugs that improve bone health.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] The Lactobacillus plantarum YS provided by this invention GuLin18 can directionally activate phenylpropane biosynthetic pathways and induce the synthesis of polyphenolic small molecule metabolites (chlorogenic acid, caffeic acid, sanguine W, etc.).

[0025] The fermentation product provided by this invention contains highly toxic alkaloids (dimethyl strychnine), carcinogenic / environmental pollutants (1,6-dinitropyrene, imidacloprid), and toxic terpenoids (paclitaxel X, 2...). It has a lower abundance of trace toxic and harmful components such as aminobenzothiazole, making it safer for food and drug use and suitable for pharmaceutical preparation.

[0026] The fermentation product prepared by the method provided by this invention can significantly upregulate flavonoids such as fisetin, kaempferol, and quercetin; polyphenols such as chlorogenic acid, caffeic acid, thymol, and sanguranol; alkaloids such as berberine, piperine, trigonelline, and betaine; and terpenoids such as aucubin, glycyrrhetinic acid, asiaticoside, and oleanolic acid.

[0027] Experiments on zebrafish have verified that the fermentation product of this invention can significantly promote bone formation in zebrafish, improve bone health, especially osteoporosis and osteoarthritis, by increasing the area of ​​the head bone and downregulating the expression levels of runx2a and acp5a.

[0028] The method for preparing food and medicine homologous fermentation products provided by this invention is simple, controllable, and can be mass-produced. Attached Figure Description

[0029] Figure 1 The diagram shows the ontology of differential metabolite classification between FD and D.

[0030] Figure 2 The diagram shows the ontology of differential metabolite classification in FD16 vs. D.

[0031] Figure 3 The second-layer KEGG pathway category bar chart of FD16 vs .D is shown;

[0032] Figure 4 The chemical structure of Mulberrofuran W is shown.

[0033] Figure 5 The staining of zebrafish skeletons in the CK group is shown;

[0034] Figure 6 The staining of the zebrafish skeletons in group M is shown;

[0035] Figure 7 The staining of the zebrafish skeletons in group A is shown;

[0036] Figure 8 The staining of the zebrafish skeletons in group B is shown;

[0037] Figure 9 The staining of the zebrafish skeletons in group C is shown;

[0038] Figure 10 The graph shows quantitative data of zebrafish skeletal staining from different groups.

[0039] Figure 11 The effects of different groups on runx2a gene expression were shown;

[0040] Figure 12 The effects of different groups on acp5a gene expression were shown. Detailed Implementation

[0041] Example 1

[0042] The screening process for probiotics with high antioxidant activity is as follows:

[0043] In this embodiment, strains with good probiotic properties (acid resistance, bile salt resistance, adhesion, drug sensitivity, etc.) were pre-screened from the longevity elderly lactic acid bacteria strain resource bank of Hunan Nutrition Tree Biotechnology Co., Ltd. These strains are BS906M7, BS88T4, BS11M9, BS619M14, BS683M16, and YS. GuLin18. Simultaneously, it assesses in vivo enzymatic antioxidants (SOD, CAT), in vitro free radical scavenging rate (DPPH), and overall antioxidant levels (T). The antioxidant properties of the fermentation supernatant of the strain were evaluated using three dimensions: AOC (autooxidative stress, organic oxidation, and organic oxidation).

[0044] SOD (total superoxide dismutase) is an important antioxidant enzyme in organisms and one of the common active ingredients in the supernatant of bacterial strains. It can specifically scavenge superoxide anion free radicals.

[0045] CAT (catalase) is another key antioxidant enzyme that catalyzes the breakdown of H2O2 into harmless water (H2O) and oxygen (O2), preventing H2O2 from accumulating in the body and transforming into more toxic hydroxyl radicals.

[0046] DPPH is a synthetically produced stable free radical (purple in color) and is a commonly used reagent for in vitro testing of the free radical scavenging ability of fermentation supernatants.

[0047] T AOC (Total Antioxidant Capacity) is an indicator that comprehensively measures the overall antioxidant capacity of all antioxidant substances (enzymes + non-enzymes) in the fermentation supernatant of a bacterial strain, rather than targeting a specific free radical or enzyme.

[0048] The bacterial culture of the above-mentioned bacteria was adjusted to a concentration of 1×10⁻⁶. 9 Centrifuge at 4℃ and 6000 rpm for 5 min, and collect the supernatant as fermentation supernatant (CFS). The antioxidant properties of the fermentation supernatant are then determined according to the above indicators.

[0049] Table 1 Antioxidant capacity of different strains

[0050]

[0051] As shown in Table 1, strain YS exhibits superior antioxidant activity in vivo, in vitro free radical scavenging, and overall antioxidant capacity. GuLin18 performed best. Given that this strain exhibited the best antioxidant activity, YS was selected. GuLin18 underwent further testing.

[0052] Strain YS The identification of GuLin18 is as follows:

[0053] Strain YS GuLin18 is a strain screened from the intestines of long-lived elderly people.

[0054] For strain YS GuLin18 was identified as *Lactobacillus plantarum* after identification.

[0055] The plant lactobacillus YS The 16S sequence of GuLin18 is as follows:

[0056]

[0057] Example 2

[0058] Fermentation raw material screening

[0059] To screen for compound ingredients with strong antioxidant properties and the ability to improve bone health, especially joint health, this experiment selected ingredients with good sales performance in domestic and international markets, as shown in Table 2. The total antioxidant capacity (T) of the ingredients was also measured. AOC), with a raw material concentration of 0.1 g / mL.

[0060] Table 2 Total antioxidant capacity of different raw materials (T) AOC)

[0061]

[0062] As shown in Table 2, T Using an AOC level of ≥30 μmol / g as the standard, high-quality food and medicinal raw materials were selected: Eucommia ulmoides leaves, Dendrobium officinale, Rehmannia glutinosa, and dried ginger were chosen as preferred raw materials. Based on these raw materials, a pharmacopoeia was consulted, revealing that these four materials are similar to the components of Eucommia ulmoides pills in Qianjin Fang (a traditional Chinese medicine formula). Therefore, these four substances were combined and fermented.

[0063] The formula uses a combination of Eucommia ulmoides leaves, Dendrobium officinale, Rehmannia glutinosa, and dried ginger, following the principle of "principal, assistant, adjuvant, and guide": [Primary herb] Eucommia ulmoides leaves (longevity leaves) are primarily used for anti-aging, and can nourish the liver and kidneys, and strengthen muscles and bones; its polyphenols (chlorogenic acid) have antioxidant properties and can protect the bone microenvironment, while its flavonoids can regulate bone metabolism pathways (bone resorption and bone formation); [Assistant Medicine] Dendrobium officinale is primarily nourishing, capable of nourishing yin and clearing heat, benefiting the stomach and promoting the production of body fluids; flavonoids can promote bone differentiation and block bone resorption pathways, while polysaccharides can maintain the bone health barrier; [Adjuvant herb] [Rehmannia glutinosa] primarily nourishes Yin, possessing cooling and blood-clearing effects, as well as the ability to nourish Yin and generate fluids; polysaccharides can act as antioxidants and promote metabolism to strengthen bone health; [Guiding herb] Dried ginger promotes digestion and absorption, warms the middle jiao (spleen and stomach), dispels cold, restores yang, and unblocks the meridians. It can also improve the digestive function of the spleen and stomach, aiding in the absorption of nourishing components and allowing the polyphenols, flavonoids, and polysaccharides in Eucommia ulmoides leaves, Dendrobium nobile, and Rehmannia glutinosa to better exert their "bone-protecting" effects, thus achieving overall "bone health." Qi and Blood Spleen and Stomach It provides holistic care for metabolism, helping to relieve bone and joint pain.

[0064] Example 3

[0065] Preparation of food and medicine homologous fermentation products

[0066] This embodiment uses YS GuLin18 fermented the above-selected raw materials (Eucommia ulmoides leaves, Dendrobium officinale, Rehmannia glutinosa, and dried ginger) and compared the changes in their active ingredients before and after fermentation.

[0067] The specific preparation process is as follows:

[0068] ① Seed liquid preparation:

[0069] YS After streaking a GuLin18 glycerol tube, pick a single bacterium and culture it in MRS liquid medium. Then, inoculate 2% bacterial culture into the MRS liquid medium and adjust the seed culture concentration to 1×10⁻⁶. 9 CFU / mL. The MRS liquid culture medium was purchased from Guangdong Huankai Microbial Technology Co., Ltd.

[0070] ② Preparation of unfermented composition:

[0071] A mixture of 5% glucose, 1% peptone, 0.25% dipotassium hydrogen phosphate, 8% food-medicine homologous composition, and water was sterilized at 115°C for 20 minutes to obtain an unfermented composition. The food-medicine homologous composition comprises 70 wt% Eucommia ulmoides leaf powder, 15 wt% Dendrobium officinale powder, 10 wt% Rehmannia glutinosa powder, and 5 wt% dried ginger powder.

[0072] ③ Seed liquid inoculation:

[0073] Inoculate the unfermented composition with YS GuLin18 seed culture, with an inoculum size of 2%;

[0074] ④ Preparation of fermentation products:

[0075] Low temperature shallow layer Fermentation is carried out using an enzymatic hydrolysis coupled targeted fermentation method, with the following specific steps:

[0076] Early stage of S1 (0 6h): Add β to the unfermented composition of the inoculated seed culture. Dextran hydrolase (enzyme activity 20 U / g), at 30 Fermentation is carried out at 32℃.

[0077] This stage consists of "substrate pretreatment + initial strain adaptation": β Dextran hydrolase can degrade difficult-to-utilize polysaccharides and polyphenols in substrates, reducing substrate complexity and facilitating rapid utilization by the strain; 30 32℃ is the suitable temperature for the initial proliferation of inoculated strains, which can help the strains quickly adapt to the system and accumulate population, laying the foundation for subsequent high-activity metabolism.

[0078] S2 Mid-term (6) 8h): Add 0.1% Vitamin C and bring to 35°C. Ferment at 37℃.

[0079] This stage is "metabolic activation + optimal temperature matching for enzyme production": 35 37℃ is the optimal temperature for the bacterial strain to produce enzymes, which can maximize the activation of its metabolic pathways and promote enzyme synthesis; Vitamin C is a color protectant that can preserve the color of food and medicinal substances and enhance the catalytic efficiency of enzymes, providing conditions for the subsequent conversion of active ingredients.

[0080] S3 late stage (8) 16h): Add 0.05% quinic acid, 35 Fermentation was carried out at 37°C to obtain a fermentation broth. The purpose of this step was to promote the conversion of caffeic acid to chlorogenic acid in the fermentation product.

[0081] This stage involves "targeted conversion + fermentation rhythm control": quinic acid can directionally regulate metabolic pathways, promoting the conversion of caffeic acid to chlorogenic acid (achieving enrichment of active ingredients); using the above low-temperature shallow fermentation... Enzymatic hydrolysis coupled with targeted fermentation can complete the conversion of active ingredients while avoiding excessive consumption of active ingredients by the strain.

[0082] The fermentation speed was controlled at 130 r / min throughout the entire fermentation process, and the final pH value was 3.8. Fermentation ends when the corresponding fermentation time and endpoint pH are reached (4.0).

[0083] ⑤ The above fermentation broth was sterilized at 115℃ for 30 min to obtain an inactivated fermentation broth. The inactivated fermentation broth was centrifuged at 6000 r / min for 5 min. The supernatant (i.e., food and medicine homologous fermentation product) was obtained by centrifugation.

[0084] Comparative Example 1

[0085] The difference from Example 1 is that a conventional fermentation process was used, with the following fermentation conditions: the temperature was maintained at 35°C throughout the process. The fermentation was carried out at 37℃, with a rotation speed of 130 r / min, for 16 hours, and the final pH value was 3.8. 4.0. Fermentation was terminated after reaching the corresponding fermentation time and endpoint pH. The fermentation broth was sterilized at 115℃ for 30 minutes to obtain an inactivated fermentation broth. The inactivated fermentation broth was centrifuged at 6000 r / min for 5 minutes. The supernatant was obtained by centrifugation.

[0086] Comparative Example 2

[0087] The unfermented composition prepared according to step 2 of Example 3 was formulated and inactivated using 5% glucose, 1% peptone, 0.25% dipotassium hydrogen phosphate, and 8% a food-medicinal homologous composition (in a ratio of 70wt%:15wt%:10wt%:5wt% for Eucommia ulmoides leaf powder: Dendrobium officinale powder: Rehmannia glutinosa powder: dried ginger powder). The inactivation temperature was 115℃ for 30 min. The inactivated fermentation broth was centrifuged at 6000 r / min for 5 min. The supernatant was obtained by centrifugation.

[0088] The present invention collected the fermentation broth (i.e., the supernatant obtained by centrifugation, labeled as FD16) prepared in Example 3; the fermentation broth (i.e., the supernatant obtained by centrifugation, labeled as FD) prepared in Comparative Example 1 and the unfermented composition (i.e., the supernatant obtained by centrifugation, labeled as D) prepared in Comparative Example 2 and conducted polyphenol, flavonoid, antioxidant capacity and non-targeted omics determination.

[0089] The relevant indicators were measured as follows:

[0090] 1. Polyphenol, flavonoid, and antioxidant capacity detection: According to the results in Table 3, the kit method showed that, compared with group D and group FD, the low-temperature shallow layer method was more effective. The fermentation broth (FD16) of enzyme-coupled targeted fermentation exhibits high levels of polyphenols, flavonoids, and total antioxidant capacity (T). Both AOC and DPPH scavenging rates were significantly improved. Compared with group D, polyphenols increased by 38.28%, flavonoids increased by 7.53%, and total antioxidant (T) levels increased significantly. AOC levels increased by 67.01%, and DPPH free radical scavenging rate increased by 57.57%.

[0091] Table 3 Comparison of total phenols, flavonoids and antioxidant indicators among different groups

[0092]

[0093] 2. Non-targeted metabolomics

[0094] Changes in overall metabolites in each group of samples were analyzed using liquid chromatography-mass spectrometry (LC-MS). The OPLS model established from the experimental data... The DA model, with R² and Q² ≥ 0.5, indicates that the model is stable and reliable, and has good explanatory and predictive abilities. OPLS is then implemented. Metabolites with significant differences were screened based on DA VIP>1 and P .value<0.05, FC>2 or FC<1 / 2, and their metabolite expression changes and functional pathways were analyzed.

[0095] ① Classification of differential metabolites

[0096] The supernatants of D and FD were compared (FD vs. D), and 15 classes of significantly different metabolites were detected. These mainly included amino acids and their derivatives (7, 2.33%), carboxylic acids and their derivatives (8, 2.66%), dipeptides (6, 2.00%), fatty acyl groups (6, 2.00%), and flavonoids. Glycosides (4, 1.33%), glycerophospholipids (4, 1.33%), glycosyl compounds (5, 1.66%), long-chain fatty acids (6, 1.99%), other (195, 64.78%), peptides (28, 9.33%), proberberine alkaloids and their derivatives (4, 1.33%), steroids and their derivatives (5, 1.66%), triterpenoid glycosides (4, 1.33%), triterpenoids (12, 3.99%) (details as follows) Figure 1 (As shown).

[0097] Comparison of the supernatants of D and FD16 (FD16 vs. D) revealed 15 significantly different metabolites. These mainly included alkaloids and their derivatives (6, 0.9%), amino acids and their derivatives (23, 3.47%), carboxylic acids and their derivatives (13, 1.96%), fatty acyl groups (13, 1.96%), and flavonoids. Glycosides (14, 2.11%), glycosyl compounds (11, 1.66%), organooxygen compounds (7, 1.06%), other (478, 72.1%), peptides (38, 5.73%), phosphatidylcholine derivatives (9, 1.36%), purine nucleosides (7, 1.06%), steroids and their derivatives (8, 1.21%), triterpenoid glycosides (7, 1.06%), triterpenoids (19, 2.87%) (details as follows) Figure 2 (As shown).

[0098] This shows that the FD16 group produced more types of metabolites than the FD group, especially flavonoid O. Glycosides and triterpenoid metabolites were abundant and accounted for a high proportion. In addition, unique alkaloids and their derivatives were also found in the FD16 group.

[0099] ② Differential metabolites

[0100] Different types of metabolites were observed after fermentation. We focused on the changes in flavonoids, polyphenols, alkaloids, and terpenoids in FD16 and FD fermentation products.

[0101] For FD vs .D, only vitexin is present after fermentation. 2 O Rhamnose, piperine, and oleanolic acid showed significant upregulation, while other substances showed no significant upregulation.

[0102] For FD16 vs FD16, fermentation significantly upregulates flavonoids such as fisetin, kaempferol, and quercetin; polyphenols such as chlorogenic acid, caffeic acid, thymol, and sanguranol; alkaloids such as berberine, piperine, trigonelline, and betaine; and terpenoids such as aucubin, glycyrrhetinic acid, asiaticoside, and oleanolic acid.

[0103] FD16 Low-Temperature Shallow Layer of this Application Following enzymatic hydrolysis coupled with targeted fermentation, sangfuran W appeared in the metabolites at high abundance. Literature review indicates that sangfuran W is not an inherent component of the raw materials (Eucommia ulmoides leaves, Rehmannia glutinosa, dried ginger, and Dendrobium officinale), but rather a unique new substance generated through synergistic effects of the raw materials and microbial enzyme catalysis. Its structural formula is shown below. Figure 4 As shown. Other typical furan components include furanocoumarins such as psoralen, and morularans, especially morularan W, which has significant antiviral, anti-inflammatory, and osteoblast differentiation-promoting effects.

[0104] Therefore, it can be inferred that FD16 can effectively decompose macromolecules and release a greater number of significantly smaller molecules.

[0105] In addition, these small molecules have a certain positive correlation with bone health. Among them, flavonoids (quercetin, fisetin, kaempferol), polyphenols (chlorogenic acid, sanguine W), alkaloids (berberine), and terpenes (glycyrrhetinic acid, aucubin) can inhibit joint inflammation and relieve synovial congestion and swelling. Quercetin, fisetin, sanguine W, aucubin, and chlorogenic acid can reduce oxidative stress, promote chondrocyte proliferation and metabolism, delay chondrocyte apoptosis, and promote cartilage repair. Plumbagoline, glycyrrhetinic acid, and piperine can reduce inflammation-mediated pain sensitivity and relieve pain during joint movement. Therefore, subsequent experiments will be conducted to verify the efficacy of the fermentation broth in promoting bone health.

[0106] Table 4. Changes in beneficial metabolites among different groups

[0107]

[0108] Note: No significant difference: There was no significant difference in the active ingredient between the FD.vs.D group (P>0.05); Increase: The active ingredient was significantly upregulated between the FD16.vs.D or FD.vs.D group (P<0.05); Decrease: The active ingredient was significantly downregulated between the FD16.vs.D or FD.vs.D group (P<0.05).

[0109] ② Significantly altered metabolic pathways after fermentation of beneficial substances: Regarding significantly altered metabolic pathways, the focus is on the biosynthesis of secondary metabolites. Figure 3 The following are the metabolic pathways that show significant changes in FD16 vs. D, including flavonoid biosynthesis, flavonone and flavonol biosynthesis, and phenylpropanoid biosynthesis. Figure 3 However, the phenylpropane biosynthetic pathway in FD vs. D did not show significant changes. This suggests that through low-temperature shallow layers... Following the enzymatic coupling targeted fermentation process, FD16 may have activated the phenylpropanoid biosynthetic pathway, leading to the significant generation of chlorogenic acid, caffeic acid, and sanguran W. Chlorogenic acid, a core component of Eucommia ulmoides leaves, is chemically caffeoylquinic acid, a key product of phenylpropanoid metabolism. It is derived from phenylpropanoid precursors (caffeoyl... CoA is formed by the ester bond between quinic acid and phenylpropanoids, and is a typical metabolite of the phenylpropanoid pathway. In summary, low-temperature shallow infiltration is employed... Enzymatic hydrolysis coupled targeted fermentation process, Lactobacillus plantarum YS GuLin18, after fermentation of Eucommia ulmoides and Rehmannia glutinosa, can directionally activate the phenylpropane biosynthetic pathway and induce the synthesis of polyphenolic small molecule metabolites (chlorogenic acid, caffeic acid, etc.).

[0110] Table 5 Significant pathways of change in beneficial substances

[0111]

[0112] Note: Yes: There is a significant upregulated metabolic pathway between the FD16 vs. D or FD vs. D groups (P < 0.05); No: There is no significant change in metabolic pathways between the FD16 vs. D or FD vs. D groups.

[0113] ④ Reduced levels of toxic and harmful components

[0114] In the fermentation process of food-medicine homologous formulations, the trace amounts of toxic and harmful components originally contained therein can be significantly degraded, especially after low-temperature shallow fermentation. After fermentation using the enzymatically coupled targeted fermentation process (FD16 vs. D), the fermentation broth contained highly toxic alkaloids (dimethyl strychnine), carcinogenic / environmentally friendly pollutants (1,6-dinitropyrene, imidacloprid), and toxic terpenoids (paclitaxel X, 2...). (Aminobenzothiazole) can significantly reduce [the concentration of aminobenzothiazole].

[0115] Table 6. Significantly Downgraded Toxic and Harmful Components

[0116]

[0117] Increased: Components were significantly upregulated in both the FD16 vs. D and FD vs. D groups (P < 0.05); Decreased: Components were significantly downregulated in both the FD16 vs. D and FD vs. D groups (P < 0.05). To further verify the relationship between fermentation products and bone health, related in vivo validation experiments were conducted in zebrafish.

[0118] Example 4

[0119] The fermentation broth of the composition promotes bone formation in zebrafish.

[0120] Healthy 4dpf juvenile fish were divided into six groups: control group (CK group), model group (M group), and sample group (A: conventional fermentation supernatant lyophilized powder, 62.5 ug / mL; B: low temperature shallow layer). Lyophilized supernatant from enzymatically coupled targeted fermentation was collected at a concentration of 62.5 μg / ml. Composition groups (62.5 μg / ml, prepared according to a 70 wt%: 15 wt%: 10 wt%: 5 wt% ratio of Eucommia ulmoides leaf powder: Dendrobium officinale powder: Rehmannia glutinosa powder: dried ginger powder) consisted of 10 samples per group. The mixture was continuously incubated at 28.5℃. A total of 4 mL of the solution was administered via exposure. The drug delivery system is shown in Table 7.

[0121] Table 7. Drug administration system (unit: mL)

[0122]

[0123] Dosing cycle: From 4 dpf to 9 dpf, administer for 5 days, changing the incubation medium containing the drug daily at a 1 / 2 ratio (2 mL). The yolk sac in zebrafish juveniles contains abundant nutrients, which can sustain the juveniles until 9 days of age. 10 dpf, therefore no feeding is required during the experiment.

[0124] Feed. The zebrafish juveniles were stained with alizarin red, and the staining results of the skull bones were observed and photographed under a biological microscope, as shown in the figure.

[0125] 5 As shown in Figure 9.

[0126] Compared with the control group (CK), the model group (M) showed blurred skull imaging and significantly underdeveloped spine, proving the modeling was successful. Five clear images of zebrafish with good posture were taken from each group and analyzed using software, such as... Figure 10 As shown.

[0127] Comparing CK and M, the quantitative data showed significant differences, indicating successful modeling. Group B demonstrated the best effect in improving bone health, significantly promoting bone formation in zebrafish. In terms of bone area, Group B was significantly superior to Groups C and A.

[0128] ②RT PCR detection of related gene expression

[0129] The specific testing method is as follows:

[0130] RNA was extracted from zebrafish brain tissue using Trizol reagent. Total RNA was reverse transcribed into cDNA using a reverse transcription kit. The cDNA was then processed using a SYBR premix Taq kit in Real-time. The reaction was performed on a time-lapse PCR instrument. Follow the procedure... The relative mRNA expression levels of relevant genes in zebrafish were calculated using the ΔΔCt method. Primers were designed, and β-type primers were selected. Actin is used as an internal reference.

[0131] runx2a is a transcription factor that plays a crucial role in bone development and bone metabolism. It is a subtype of the runx2 gene, which plays a vital role in bone formation, especially in the differentiation and maturation of osteoblasts.

[0132] ACP5A is an important biomarker in the development of osteoporosis and osteoarthritis. Its role is focused on promoting bone resorption and it is used in clinical practice as an important indicator for assessing bone metabolism.

[0133] Depend on Figure 11 and Figure 12 It can be seen that after modeling, the gene expression levels of runx2a and acp5a increased, and their expression levels decreased after different group treatments. In particular, the expression levels of runx2a and acp5a in group B could be restored to normal.

[0134] In summary, after low temperature shallow layer After enzymatic decoupling and targeted fermentation, the resulting ferments have a significant effect on improving bone health, including osteoarthritis and osteoporosis. This is mainly reflected in increasing bone surface area, downregulating the gene expression of runx2a and acp5a, and restoring normal bone health levels.

Claims

1. A type of *Lactobacillus plantarum* YS-GuLin18, characterized in that: Its accession number is CGMCC NO. 35529, and its Latin name is Lactiplantibacillus plantarum.

2. A fermented product of gentian, characterized in that: The food and medicine homologous composition was obtained by fermentation of *Lactobacillus plantarum* YS-GuLin18 as described in claim 1; the food and medicine homologous composition is composed of 70wt% *Eucommia ulmoides* leaves, 15wt% *Rehmannia glutinosa*, 10wt% dried ginger, and 5wt% *Dendrobium officinale*.

3. The method for preparing the fermented product of *Rhizoma Cyrtomegaloides* as described in claim 2, characterized in that: Includes the following steps: Seed culture of *Lactobacillus plantarum* YS-GuLin18 was inoculated into a sterilized substrate consisting of glucose, peptone, dipotassium hydrogen phosphate, a food-medicine homologous composition, and water. β-glucan hydrolase was then added to the substrate for the first fermentation step, followed by the addition of vitamin C for the second fermentation step, and then the addition of quinic acid for the third fermentation step. Finally, the substrate was inactivated, centrifuged, and the supernatant was collected.

4. The method for preparing the fermented product of *Rhizoma Cyrtomegaloides* as described in claim 3, characterized in that: The method for preparing the seed culture includes the following steps: *Lactobacillus plantarum* YS-GuLin18 is inoculated into MRS liquid medium for culture to obtain a bacterial suspension; then, 2% of the bacterial suspension is inoculated into the MRS liquid medium to obtain the seed culture; the concentration of *Lactobacillus plantarum* YS-GuLin18 in the seed culture is 1×10⁻⁶. 9 CFU / mL.

5. The method for preparing the fermented product of *Rhizoma Cylindricae* as described in claim 3, characterized in that: The substrate comprises 4-6% glucose, 0.5-1% peptone, 0.10-0.25% dipotassium hydrogen phosphate, and 6-8% food and drug homologous composition.

6. The method for preparing the fermented product of *Rhizoma Cyrtomegaloides* as described in claim 3, characterized in that: The sterilization temperature is 115℃ and the time is 20 min; the inoculation amount of the seed liquid is 2%.

7. The method for preparing the fermented product of *Rhizoma Cyrtomegaloides* as described in claim 3, characterized in that: The β-glucan hydrolase has an enzyme activity of 20 U / g, and the fermentation temperature of the first fermentation step is 30-32℃; the amount of vitamin C added is 0.1%, and the fermentation temperature of the second fermentation step is 35-37℃; the amount of quinic acid added is 0.05%, and the fermentation temperature of the third fermentation step is 35-37℃.

8. The method for preparing the fermented product of *Rhizoma Cyrtomegaloides* as described in claim 3, characterized in that: The final pH of the third fermentation step is 3.8-4.0; the inactivation temperature is 115℃ and the time is 30 min; the centrifugation speed is 6000 r / min and the time is 3-5 min.

9. The application of the fermented product of *Rhizoma Cyrtomegaloides* as described in claim 2, characterized in that: Preparation of drugs used to improve osteoporosis.

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